THE CD134/CD134L PATHWAY IS CRITICAL IN CD8-MEDIATED COSTIMULATION BLOCKADE RESISTANT REJECTION.

Abstract

O460* Intro: Two of the best characterized costimulatory pathways include CD28/B7 and CD40/CD154. Blockade of these pathways simultaneously, using fusion protein constructs (eg. CTLA4-Ig) and/or mAbs, can promote prolonged allograft survival. Interestingly there are some models in which costimulation blockade is less effective, primarily due to the ability of CD8 T cells to act in a CD28/CD154 independent manner. In recent years numerous novel costimulatory pathways have been described. In the current study we tested whether signaling through any of these alternative costimulatory pathways could be critical in CD28/CD154 blockade resistant rejection. Methods: GvHD model-recipient mice were lethally irradiated (1800 rads) after which ∼2x107 T cells were adoptively transferred intravenously. Recipient mice were then treated with the various combinations of reagents (as described below) on days 0 and 2. Mice were then sacrificed at 72 hours post-transfer and splenocytes were analyzed by flow cytometry to calculate the number of transferred cells undergoing division. Skin graft model C57BL/6, C57BL/6 IFNγ−/−, or C57BL/6 IFNγR−/− mice were used as recipients and BALB/c mice as donors. CTLA4-Ig, anti-CD40L, anti-CD134L, anti-ICOSL, anti-CD70, anti-41BBL, & anti-TRANCE were given days 0,2,4,6 (500ug i.p.). Results: For the initial evaluation we employed a well-described GvHD model. Irradiated BALB/c mice received B6 T cells which had previously been labeled with CFSE to track cellular division in-vivo. Experimental groups included animals which received no treatment, costimulation blockade (CoB) alone (CTLA4-Ig/anti-CD154) or CoB in combination with an additional agent. Additional agents tested included anti-ICOSL, anti-CD70, anti-41BBL, or anti-CD134L, anti-TRANCE. The combinations of CoB + anti-ICOSL (p<0.02) and CoB + anti-CD134L (p<0.03) significantly inhibited both CD4 and CD8 T cell proliferation when compared to CoB alone. In subsequent experiments we evaluated these agents in a fully allogeneic mouse skin graft model. Untreated mice rejected their skin graft promptly (MST 11 days). Animals treated with CB alone demonstrated a slight prolongation (MST 20 days). Despite inhibiting T cell proliferation in the GvHD model the addition of anti-ICOSL only modestly prolonged skin graft survival when compared to CB alone (MST 30 days). The addition of anti-CD70, anti-41BBL, or anti-TRANCE did not significantly prolong survival (MST 22, 23, 21 days respectively). In contrast the combination of anti-CD134L and CoB dramatically prolonged fully disparate skin allografts (MST 130 days). Further experiments defined a critical role for IFNγ. The addition of anti-IFNγ mAb or the use of IFNγ −/− mice abrogated the beneficial effects of the combined CoB/anti-CD134L treatment. Treatment of animals reconstituted with CD8 T cells from IFNγR−/− mice results in early rejection (MST =28 days vs >54 days in wt animals w/o reconstitution) suggesting that one of the effects of IFNγ is directly on CD8 T cells. Further studies aim to identify the role of CD134/CD134L interactions in CD8 T cell activation. Conclusions: The CD134/CD134L pathway plays a critical role in CD8-mediated costimulation blockade resistant rejection. Identification of key costimulatory pathways may allow for the development of potent, novel immunosuppressive therapies that are highly specific to the immune system thereby eliminating many of the unwanted side effects associated with current conventional immunosuppression.